The level of neurodegeneration was further studied by determining the plasma levels of S100β, a marker of neuronal injury, in P7 rats exposed to 1.5% ISO for 6 hours, with and without ISO PC (Fig. 3). The ELISA assay did not reveal significant differences in plasma S100β levels between these groups (Tables 1 and 2).
The effect of PC on the apoptotic pathway was also examined by caspase-12 activation in the developing brain. Western blot analysis of caspase-12 levels in the P7 cerebral cortex (Fig. 4A) after either a 6-hour exposure to 1.5% ISO or PC with ISO before the prolonged exposure were not significantly different from controls (Fig. 4B; Tables 1 and 2).
Autophagy, after anesthetic exposures, with and without PC, was examined in the P7 developing brain by measuring Beclin-1 levels. Western blot analysis (Fig. 5A) showed that a 6-hour ISO exposure did not significantly change Beclin-1 levels (Fig. 5B; Tables 1 and 2).
This study provides new evidence that PC with ISO, before a long ISO exposure, significantly decreases ISO-mediated apoptosis in the developing brain. This finding is based on a significant reduction in the caspase-3 levels using Western blot assays. However, significant differences were not detected in caspase-3 levels in the cerebral cortex and hippocampus after either ISO exposure or PC. Other markers of neuronal injury, S100β, caspase-12, and Beclin-1, were not significantly affected by either the prolonged ISO exposure or PC. While we have been able to exclude a large effect of these markers, it is possible that we could not detect significant effects due to our small sample size (e.g., Figs. 2 and 3).
Caspase-3 is one of the final mediators of the apoptotic pathway and is a well-established biomarker of apoptosis. The most important aspect of our results is that ISO PC decreased caspase-3 levels induced by a prolonged ISO exposure in the developing brain. In a similar study, Shu et al26 showed that xenon pretreatment before a combined ISO/nitrous oxide exposure decreased apoptosis, while nitrous oxide PC had no effect. Furthermore, we have previously shown that sevoflurane PC can also inhibit neuronal cell death induced by prolonged exposure to ISO.2 Thus, more studies are necessary to determine the mechanism for the protective effect of PC so that novel approaches can be developed to mimic this effect.
Anesthetics have been shown to be both neuroprotective and neurotoxic in the developing brain, and thus concentrations and durations must be considered in pediatric anesthetic practice. Our previous study suggested that prolonged exposure to sevoflurane can induce neuronal damage in vitro.27 The IV anesthetic, propofol, has also been shown to be both neurotoxic28,29 and neuroprotective against brain damage induced by ischemia and other stress factors.30,31 Therefore, it is important to investigate the dose and time responses of anesthetic-induced effects in the developing brain to use their neuroprotective features but minimize their neurotoxic effects.
S100β, a dimeric cytosolic calcium-binding protein released by glial cells, is a biomarker of blood–brain barrier dysfunction32 and overall brain distress.33 It has been studied clinically as a biomarker for traumatic brain injury and hypoxic-ischemic brain injury.34,35 We have previously studied S100β in the developing fetal rat brain and found that an in utero exposure to 3% ISO for 1 hour resulted in higher levels of S100β in the plasma of fetal rats when compared with controls.25 Furthermore, we showed an increase in plasma S100β after exposure to a subclinical concentration of ISO in neonatal mice.15 Though the current study did not show a significant difference in S100β, S100β may be a useful biomarker to detect anesthetic-mediated damage in the developing brain as indicated above, although further studies are needed to investigate its role in pediatric patients.
Caspase-12, part of the apoptotic pathway, is activated by disruption of the calcium homeostasis in the endoplasmic reticulum (ER).36 Anesthetics have been shown to cause calcium dysregulation in the ER via multiple mechanisms.37 In immature hippocampal neurons, ISO exposure was shown to enhance γ-aminobutyric acid–induced intracellular calcium increase, which was blocked by dantrolene, indicating that ISO exposure causes ryanodine receptor–dependent calcium release from the ER,17 which is consistent with our previous studies in different types of neurons.5 Other studies also suggest that ISO exposure during brain development causes increased activation of inositol triphosphate receptors (InsP3R), resulting in increased calcium release from the ER leading to cell damage and neurodegeneration.3,4,27,38 Furthermore, caspase-12–positive neurons in the hippocampus were significantly increased in fetal rats exposed to 1.3% ISO for 4 hours.39 Caspase-12 has also been shown to indirectly activate caspase-3 in the neuronal apoptotic pathway.40 Although a previous study suggested that ISO-induced neuroapoptosis during brain development in rodents involved both intrinsic and extrinsic pathways,41 it is not clear whether the caspase-12–dependent pathway is also involved. In this study, we did not find significant caspase-12 activation after the ISO exposures, possibly because our ISO concentration may not have been high enough to cause ER stress and caspase-12–dependent neuroapoptosis. Further dose-dependent and exposure duration studies are needed to clarify this question.
Beclin-1, a protein required for autophagosome formation, is an important regulator and biomarker of autophagy activity.42–44 Interestingly, autophagy may have both beneficial and harmful effects on the brain, depending on the experimental conditions. Autophagy appears to be essential to both ischemic and hyperbaric oxygen PC before cerebral ischemia.45 Furthermore, reducing Beclin-1 levels has been shown to exacerbate neurodegeneration in Alzheimer disease models, while overexpression of Beclin-1 can prevent neuronal cell death.46 Autophagy activity can be regulated by InsP3R activity, while ISO has been shown to activate InsP3R and cause cell apoptosis by overactivation of InsP3R.47,48 In addition, autophagy activity may be an upstream regulator of apoptosis, and excessive autophagy may lead to cell death by apoptosis. The effects of InsP3R activity on autophagy depend on the level of InsP3R activation, which then determines whether the effect will be protective or toxic. While this study did not find an effect on Beclin-1 expression at P7 after exposure to 1 concentration of ISO, further studies are needed to investigate the effects of general anesthetics on cell autophagy, and therefore neuroprotection or neurotoxicity, especially in the developing brain.
While we have not yet discovered the mechanism by which ISO PC prevents ISO-induced apoptosis, other groups have studied ISO PC before cerebral ischemia and have linked several mechanisms to this process. One study found that ISO PC caused a decrease in glutamate receptor activation,49 and another found that it decreased protein aggregation.50 Changes in the expression of various genes have also been discovered, but the significance of these genetic changes has not been fully determined.51–53 Furthermore, ISO may provide PC neuroprotection by causing a moderate increase of cytosolic calcium concentrations via adequate activation of the InsP3R calcium channel.54,55 While we recognize that ISO PC for brain ischemia is not the same as for a subsequent anesthetic exposure, it is possible that there may be similarities in the cascade of events and consequences. One of the limitations of the current study is that we only investigated a few of the potential mechanisms underlying the dual effects of neuroprotection and neurotoxicity caused by ISO. Future experiments could continue to address the many other mechanisms that are likely involved in this process.
The long-term behavioral adaptations to the effects of preadolescent drug exposures are more permanent compared with the same exposures later in life,56 with the peak period of anesthetic-induced apoptosis occurring during synaptogenesis.57 This vulnerable period during rat development is between approximately postnatal days 2 and 14 and between postconception day 153 and postnatal day 288 in the human, based on a species prediction model recently developed to correlate the timing of neural events between species.58 Translating exact developmental milestones between rats and humans is complicated. Postnatal brain maturation in the rat encompasses many developmental events, such as neurogenesis, neuronal migration, synaptogenesis, and apoptosis, the extent of which varies greatly, depending on the brain region of interest.59,60
The present study tested the hypothesis that PC with ISO can prevent apoptosis caused by a prolonged exposure to ISO, and our results with caspase-3, but not other markers of neuronal injury, support this hypothesis and indicate that in vivo ISO PC is neuroprotective, while a prolonged exposure to ISO is neurotoxic during early postnatal brain development. It is important to determine the optimal concentration and duration range for anesthetic exposures during postnatal brain development, which has implications for our pediatric patients.
The authors would like to thank Rebecca Speck, PhD, MPH, Department of Anesthesiology and Critical Care, University of Pennsylvania, for advice and assistance with the statistical analyses.
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